Vesicular HMGB1 release from neurons stressed with spreading depolarization enables confined inflammatory signaling to astrocytes.

The role of high mobility group box 1 (HMGB1) in inflammation is well characterized in the immune system and in response to tissue injury. More recently, HMGB1 was also shown to initiate an "inflammatory signaling cascade" in the brain parenchyma after a mild and brief disturbance, such as cortical spreading depolarization (CSD), leading to headache. Despite substantial evidence implying a role for inflammatory signaling in prevalent neuropsychiatric disorders such as migraine and depression, how HMGB1 is released from healthy neurons and how inflammatory signaling is initiated in the absence of apparent cell injury are not well characterized. We triggered a single cortical spreading depolarization by optogenetic stimulation or pinprick in naïve Swiss albino or transgenic Thy1-ChR2-YFP and hGFAP-GFP adult mice. We evaluated HMGB1 release in brain tissue sections prepared from these mice by immunofluorescent labeling and immunoelectron microscopy. EzColocalization and Costes thresholding algorithms were used to assess the colocalization of small extracellular vesicles (sEVs) carrying HMGB1 with astrocyte or microglia processes. sEVs were also isolated from the brain after CSD, and neuron-derived sEVs were captured by CD171 (L1CAM). sEVs were characterized with flow cytometry, scanning electron microscopy, nanoparticle tracking analysis, and Western blotting. We found that HMGB1 is released mainly within sEVs from the soma of stressed neurons, which are taken up by surrounding astrocyte processes. This creates conditions for selective communication between neurons and astrocytes bypassing microglia, as evidenced by activation of the proinflammatory transcription factor NF-ĸB p65 in astrocytes but not in microglia. Transmission immunoelectron microscopy data illustrated that HMGB1 was incorporated into sEVs through endosomal mechanisms. In conclusion, proinflammatory mediators released within sEVs can induce cell-specific inflammatory signaling in the brain without activating transmembrane receptors on other cells and causing overt inflammation.

Migraine and neuroinflammation: the inflammasome perspective.

BACKGROUND: Neuroinflammation has an important role in the pathophysiology of migraine, which is a complex neuro-glio-vascular disorder. The main aim of this review is to highlight findings of cortical spreading depolarization (CSD)-induced neuroinflammatory signaling in brain parenchyma from the inflammasome perspective. In addition, we discuss the limited data of the contribution of inflammasomes to other aspects of migraine pathophysiology, foremost the activation of the trigeminovascular system and thereby the generation of migraine pain. MAIN BODY: Inflammasomes are signaling multiprotein complexes and key components of the innate immune system. Their activation causes the production of inflammatory cytokines that can stimulate trigeminal neurons and are thus relevant to the generation of migraine pain. The contribution of inflammasome activation to pain signaling has attracted considerable attention in recent years. Nucleotide-binding domain (NOD)-like receptor family pyrin domain containing 3 (NLRP3) is the best characterized inflammasome and there is emerging evidence of its role in a variety of inflammatory pain conditions, including migraine. In this review, we discuss, from an inflammasome point of view, cortical spreading depolarization (CSD)-induced neuroinflammatory signaling in brain parenchyma, the connection with genetic factors that make the brain vulnerable to CSD, and the relation of the inflammasome with diseases that are co-morbid with migraine, including stroke, epilepsy, and the possible links with COVID-19 infection. CONCLUSION: Neuroinflammatory pathways, specifically those involving inflammasome proteins, seem promising candidates as treatment targets, and perhaps even biomarkers, in migraine.

Pericytes in Ischemic Stroke.

Recent stroke research has shifted the focus to the microvasculature from neuron-centric views. It is increasingly recognized that a successful neuroprotection is not feasible without microvascular protection. On the other hand, recent studies on pericytes, long-neglected cells on microvessels have provided insight into the regulation of microcirculation. Pericytes play an essential role in matching the metabolic demand of nervous tissue with the blood flow in addition to regulating the development and maintenance of the blood-brain barrier (BBB), leukocyte trafficking across the BBB and angiogenesis. Pericytes appears to be highly vulnerable to injury. Ischemic injury to pericytes on cerebral microvasculature unfavorably impacts the stroke-induced tissue damage and brain edema by disrupting microvascular blood flow and BBB integrity. Strongly supporting this, clinical imaging studies show that tissue reperfusion is not always obtained after recanalization. Therefore, prevention of pericyte dysfunction may improve the outcome of recanalization therapies by promoting microcirculatory reperfusion and preventing hemorrhage and edema. In the peri-infarct tissue, pericytes are detached from microvessels and promote angiogenesis and neurogenesis, and hence positively effect stroke outcome. Expectedly, we will learn more about the place of pericytes in CNS pathologies including stroke and devise approaches to treat them in the next decades.

Aura and Stroke: relationship and what we have learnt from preclinical models.

BACKGROUND: Population-based studies have highlighted a close relationship between migraine and stroke. Migraine, especially with aura, is a risk factor for both ischemic and hemorrhagic stroke. Interestingly, stroke risk is highest for migraineurs who are young and otherwise healthy. MAIN BODY: Preclinical models have provided us with possible mechanisms to explain the increased vulnerability of migraineurs' brains towards ischemia and suggest a key role for enhanced cerebral excitability and increased incidence of microembolic events. Spreading depolarization (SD), a slowly propagating wave of neuronal depolarization, is the electrophysiologic event underlying migraine aura and a known headache trigger. Increased SD susceptibility has been demonstrated in migraine animal models, including transgenic mice carrying human mutations for the migraine-associated syndrome CADASIL and familial hemiplegic migraine (type 1 and 2). Upon experimentally induced SD, these mice develop aura-like neurological symptoms, akin to patients with the respective mutations. Migraine mutant mice also exhibit an increased frequency of ischemia-triggered SDs upon experimental stroke, associated with accelerated infarct growth and worse outcomes. The severe stroke phenotype can be explained by SD-related downstream events that exacerbate the metabolic mismatch, including pericyte contraction and neuroglial inflammation. Pharmacological suppression of the genetically enhanced SD susceptibility normalizes the stroke phenotype in familial hemiplegic migraine mutant mice. Recent epidemiologic and imaging studies suggest that these preclinical findings can be extrapolated to migraine patients. Migraine patients are at risk for particularly cardioembolic stroke. At the same time, studies suggest an increased incidence of coagulopathy, atrial fibrillation and patent foramen ovale among migraineurs, providing a possible path for microembolic induction of SD and, in rare instances, stroke in hyperexcitable brains. Indeed, recent imaging studies document an accelerated infarct progression with only little potentially salvageable brain tissue in acute stroke patients with a migraine history, suggesting an increased vulnerability towards cerebral ischemia. CONCLUSION: Preclinical models suggest a key role for enhanced SD susceptibility and microembolization to explain both the occurrence of migraine attacks and the increased stroke risk in migraineurs. Therapeutic targeting of SD and microembolic events, or potential causes thereof, will be promising for treatment of aura and may also prevent ischemic infarction in vulnerable brains.

Improving Microcirculatory Reperfusion Reduces Parenchymal Oxygen Radical Formation and Provides Neuroprotection.

BACKGROUND AND PURPOSE: Reperfusion is the most significant determinant of good outcome after ischemic stroke. However, complete reperfusion often cannot be achieved, despite satisfactory recanalization. We hypothesized that microvascular protection was essential for achieving effective reperfusion and, hence, neuroprotection. To test this hypothesis, we have developed an in vivo model to differentially monitor parenchymal and vascular reactive oxygen species (ROS) formation. By comparing the ROS-suppressing effect of N-tert-butyl-α-phenylnitrone (PBN) with its blood-brain barrier impermeable analog 2-sulfo-phenyl-N-tert-butylnitrone (S-PBN), we assessed the impact of vascular ROS suppression alone on reperfusion and stroke outcome after recanalization. METHODS: The distal middle cerebral artery was occluded for 1 hour by compressing with a micropipette and then recanalized (n=60 Swiss mice). ROS formation was monitored for 1 hour after recanalization by intravital fluorescence microscopy in pial vasculature and cortical parenchyma with topically applied hydroethidine through a cranial window. PBN (100 mg/kg) or S-PBN (156 mg/kg) was administered shortly before recanalization, and suppression of the vascular and parenchymal hydroethidine fluorescence was examined (n=22). Microcirculatory patency, reperfusion, ischemic tissue size, and neurological outcome were also assessed in a separate group of mice 1 to 72 hours after recanalization (n=30). RESULTS: PBN and S-PBN completely suppressed the reperfusion-induced increase in ROS signal within vasculature. PBN readily suppressed ROS produced in parenchyma by 88%. S-PBN also suppressed the parenchymal ROS by 64% but starting 40 minutes later. Intriguingly, PBN and S-PBN comparably reduced the size of ischemic area by 65% and 48% (P>0.05), respectively. S-PBN restored the microvascular patency and perfusion after recanalization, suggesting that its delayed parenchymal antioxidant effect could be secondary to improved microcirculatory reperfusion. CONCLUSIONS: Promoting microvascular reperfusion by protecting vasculature can secondarily reduce parenchymal ROS formation and provide neuroprotection. The model presented can be used to directly assess pharmacological end points postulated in brain parenchyma and vasculature in vivo.

12/15-lipoxygenase inhibition attenuates neuroinflammation by suppressing inflammasomes.

Introduction: Lipoxygenases (LOXs) have essential roles in stroke, atherosclerosis, diabetes, and hypertension. 12/15-LOX inhibition was shown to reduce infarct size and brain edema in the acute phase of experimental stroke. However, the significance of 12/15-LOX on neuroinflammation, which has an essential role in the pathophysiology of stroke, has not been clarified yet. Methods: In this study, ischemia/recanalization (I/R) was performed by occluding the proximal middle cerebral artery (pMCAo) in mice. Either the 12/15-LOX inhibitor (ML351, 50 mg/kg) or its solvent (DMSO) was injected i.p. at recanalization after 1 h of occlusion. Mice were sacrificed at 6, 24, and 72-h after ischemia induction. Infarct volumes were calculated on Nissl-stained sections. Neurological deficit scoring was used for functional analysis. Lipid peroxidation was determined by the MDA assay, and the inflammatory cytokines IL-6, TNF-alpha, IL-1beta, IL-10, and TGF-beta were quantified by ELISA. The inflammasome proteins NLRP1 and NLRP3, 12/15-LOX, and caspase-1 were detected with immunofluorescence staining. Results: Infarct volumes, neurological deficit scores, and lipid peroxidation were significantly attenuated in ML351-treated groups at 6, 24, and 72-h. ELISA results revealed that the pro-inflammatory cytokines IL-1beta, IL-6, and TNF-alpha were significantly decreased at 6-h and/or 24-h of I/R, while the anti-inflammatory cytokines IL-10 and TNF-alpha were increased at 24-h or 72-h of ML351 treatment. NLRP1 and NLRP3 immunosignaling were enhanced at three time points after I/R, which were significantly diminished by the ML351 application. Interestingly, NLRP3 immunoreactivity was more pronounced than NLRP1. Hence, we proceeded to study the co-localization of NLRP3 immunoreactivity with 12/15-LOX and caspase-1, which indicated that NLRP3 was co-localized with 12/15-LOX and caspase-1 signaling. Additionally, NLRP3 was found in neurons at all time points but in non-neuronal cells 72 h after I/R. Discussion: These results suggest that 12/15-LOX inhibition suppresses ischemia-induced inflammation in the acute and subacute phases of stroke via suppressing inflammasome activation. Understanding the mechanisms underlying lipid peroxidation and its associated pathways, like inflammasome activation, may have broader implications for the treatment of stroke and other neurological diseases characterized by neuroinflammation.

Cortical spreading depolarization-induced constriction of penetrating arteries can cause watershed ischemia: A potential mechanism for white matter lesions.

Periventricular white matter lesions (WMLs) are common MRI findings in migraine with aura (MA). Although hemodynamic disadvantages of vascular supply to this region create vulnerability, the pathophysiological mechanisms causing WMLs are unclear. We hypothesize that prolonged oligemia, a consequence of cortical spreading depolarization (CSD) underlying migraine aura, may lead to ischemia/hypoxia at hemodynamically vulnerable watershed zones fed by long penetrating arteries (PAs). For this, we subjected mice to KCl-triggered single or multiple CSDs. We found that post-CSD oligemia was significantly deeper at medial compared to lateral cortical areas, which induced ischemic/hypoxic changes at watershed areas between the MCA/ACA, PCA/anterior choroidal and at the tip of superficial and deep PAs, as detected by histological and MRI examination of brains 2-4 weeks after CSD. BALB-C mice, in which MCA occlusion causes large infarcts due to deficient collaterals, exhibited more profound CSD-induced oligemia and were more vulnerable compared to Swiss mice such that a single CSD was sufficient to induce ischemic lesions at the tip of PAs. In conclusion, CSD-induced prolonged oligemia has potential to cause ischemic/hypoxic injury at hemodynamically vulnerable brain areas, which may be one of the mechanisms underlying WMLs located at the tip of medullary arteries seen in MA patients.

Reduced folate carrier 1 is present in retinal microvessels and crucial for the inner blood retinal barrier integrity.

BACKGROUND: Reduced folate carrier 1 (RFC1; SLC19a1) is the main responsible transporter for the B9 family of vitamins named folates, which are essential for normal tissue growth and development. While folate deficiency resulted in retinal vasculopathy, the expression and the role of RFC1 in blood-retinal barrier (BRB) are not well known. METHODS: We used whole mount retinas and trypsin digested microvessel samples of adult mice. To knockdown RFC1, we delivered RFC1-targeted short interfering RNA (RFC1-siRNA) intravitreally; while, to upregulate RFC1 we delivered lentiviral vector overexpressing RFC1. Retinal ischemia was induced 1-h by applying FeCl3 to central retinal artery. We used RT-qPCR and Western blotting to determine RFC1. Endothelium (CD31), pericytes (PDGFR-beta, CD13, NG2), tight-junctions (Occludin, Claudin-5 and ZO-1), main basal membrane protein (Collagen-4), endogenous IgG and RFC1 were determined immunohistochemically. RESULTS: Our analyses on whole mount retinas and trypsin digested microvessel samples of adult mice revealed the presence of RFC1 in the inner BRB and colocalization with endothelial cells and pericytes. Knocking down RFC1 expression via siRNA delivery resulted in the disintegration of tight junction proteins and collagen-4 in twenty-four hours, which was accompanied by significant endogenous IgG extravasation. This indicated the impairment of BRB integrity after an abrupt RFC1 decrease. Furthermore, lentiviral vector-mediated RFC1 overexpression resulted in increased tight junction proteins and collagen-4, confirming the structural role of RFC1 in the inner BRB. Acute retinal ischemia decreased collagen-4 and occludin levels and led to an increase in RFC1. Besides, the pre-ischemic overexpression of RFC1 partially rescued collagen-4 and occludin levels which would be decreased after ischemia. CONCLUSION: In conclusion, our study clarifies the presence of RFC1 protein in the inner BRB, which has recently been defined as hypoxia-immune-related gene in other tissues and offers a novel perspective of retinal RFC1. Hence, other than being a folate carrier, RFC1 is an acute regulator of the inner BRB in healthy and ischemic retinas.

Microvascular protection is essential for successful neuroprotection in stroke.

Currently, the best way of neuroprotection for acute ischemic stroke appears to be restoration of blood flow to the ischemic area by thrombolysis. Unfortunately, a short therapeutic time window as well as thrombolysis-induced bleeding and edema limit the use of recanalization therapies. Here, we review the evidence suggesting that ischemia/reperfusion-induced microvascular injury plays a critical role in determining tissue survival after recanalization in focal cerebral ischemia by disrupting the blood-brain barrier integrity and promoting microcirculatory clogging. Among many complex mechanisms of the ischemia-reperfusion injury, overproduction of oxygen and nitrogen radicals on the microvascular wall appears to significantly contribute to these pathological processes. These developments bring about the exciting possibility that effective suppression of oxidative/nitrative stress during pharmacological or interventional re-opening of the occluded artery may significantly improve the outcome of recanalization therapies in stroke patients by improving microcirculatory reflow as well as by preventing hemorrhagic conversion and vasogenic edema. They also point to the critical (but partly neglected) importance of the microcirculation in neuroprotection.

Fingolimod provides long-term protection in rodent models of cerebral ischemia.

OBJECTIVE: The sphingosine-1-phosphate (S1P) receptor agonist fingolimod (FTY720), that has shown efficacy in advanced multiple sclerosis clinical trials, decreases reperfusion injury in heart, liver, and kidney. We therefore tested the therapeutic effects of fingolimod in several rodent models of focal cerebral ischemia. To assess the translational significance of these findings, we asked whether fingolimod improved long-term behavioral outcomes, whether delayed treatment was still effective, and whether neuroprotection can be obtained in a second species. METHODS: We used rodent models of middle cerebral artery occlusion and cell-culture models of neurotoxicity and inflammation to examine the therapeutic potential and mechanisms of neuroprotection by fingolimod. RESULTS: In a transient mouse model, fingolimod reduced infarct size, neurological deficit, edema, and the number of dying cells in the core and periinfarct area. Neuroprotection was accompanied by decreased inflammation, as fingolimod-treated mice had fewer activated neutrophils, microglia/macrophages, and intercellular adhesion molecule-1 (ICAM-1)-positive blood vessels. Fingolimod-treated mice showed a smaller infarct and performed better in behavioral tests up to 15 days after ischemia. Reduced infarct was observed in a permanent model even when mice were treated 4 hours after ischemic onset. Fingolimod also decreased infarct size in a rat model of focal ischemia. Fingolimod did not protect primary neurons against glutamate excitotoxicity or hydrogen peroxide, but decreased ICAM-1 expression in brain endothelial cells stimulated by tumor necrosis factor alpha. INTERPRETATION: These findings suggest that anti-inflammatory mechanisms, and possibly vasculoprotection, rather than direct effects on neurons, underlie the beneficial effects of fingolimod after stroke. S1P receptors are a highly promising target in stroke treatment.

Importance of Pericytes in the Pathophysiology of Cerebral Ischemia.

Various cell types contribute to pathological changes observed in the brain following cerebral ischemia. Pericytes, as a component of neurovascular unit (NVU) and blood brain barrier (BBB), play a key role for cerebral blood flow control and regulation of vessel permeability. It was shown that pericytes can control cerebral blood flow at the level of capillaries, by their contractile property. Their role in BBB development and maintenance are crucial for guidance of brain vessel development, new vessel formation and stabilization of the newly formed vessels. Additionally, they can contribute to inflammation in response to inflammatory stimuli and can differentiate to various cell types by their multipotent differentiation properties. This cell type which is intimately associated with cerebral circulation also plays important roles during cerebral ischemia. Here, we review the properties and physiological functions of pericytes, how these functions change during ischemia to affect the pathophysiology of ischemic stroke and post stroke cognitive impairment. Pericytes are a neglected cell type and they are not unambiguously characterized which in turn led to contradictory findings in the literature. Clear characterization of pericytes by current methods will help better understanding of their role in the pathophysiology of stroke. With the information gained from these efforts it will be possible to develop pericyte specific therapeutic targets and achieve important breakthroughs in clinical recovery in ischemic stroke treatment.

Early Cerebral Blood Flow Changes, Cerebrovascular Reactivity and Cortical Spreading Depolarizations in Experimental Mild Traumatic Brain Injury Model.

AIM: To investigate the spatiotemporal dynamics of early cerebral blood flow (CBF) changes, cerebrovascular reactivity (CVR), and vascular responses to cortical spreading depolarization (CSD) in an experimental mild traumatic brain injury (mTBI) model with laser speckle contrast imaging (LSCI) technique. MATERIAL AND METHODS: The weight-drop model was used to induce blunt head trauma. The mice were divided into two groups as mild TBI (n=12), and sham (n=6). The animals underwent continuous LSCI before and for 1 hour after trauma to evaluate the regional CBF changes, CVR in response to CO2, and CSD-associated vascular responses induced by pinprick. RESULTS: Our minor blunt head trauma protocol induced CSD in only 2 (16.7%) animals, which were excluded from further analyses. Of the remaining animals, 30% showed slight hyperemia following trauma, with mild ipsilateral hemisphere oligemia (15%?20% decrease in CBF) on average compared to baseline (p=0.027) and contralateral hemisphere (p=0.029). Maximal CBF decrease was measured in the peri-impact area (24.1% ± 5.1%). No significant difference was found between the sham and mTBI groups and two hemispheres of the mTBI group or pre/post-CSD periods of CO2 reactivity, as well as the characteristics of vascular CSD responses (net ischemia: 52.3% ± 2.6% vs. 56.3% ± 1.9% and prolonged oligemia duration 44.8 ± 1.8 min vs. 49.8 ± 2.3 min). CONCLUSION: The ipsilateral hemisphere, particularly in the peri-impact area, had mild hypoperfusion, within the first hour of minor blunt head trauma in mice. Nonetheless, mTBI does not alter CVR and vascular responses to an induced CSD, thus the overall CVR is largely preserved in mTBI without significant structural damage despite a mildly decreased CBF in the hyperacute phase.

Brain microvascular pericytes in health and disease.

Pericytes are located at periphery of the microvessel wall and wrap it with their processes. They communicate with other cells of the neurovascular unit by direct contact or through signaling pathways and regulate several important microcirculatory functions. These include development and maintenance of the blood-brain barrier (BBB), distribution of the capillary blood flow to match the local metabolic need of the nearby cells, and angiogenesis. Pericytes also exhibit phagocytic activity and may function as pluripotent stem cells. Increasing evidence suggests a role for pericytes in a wide range of CNS diseases. They appear to be vulnerable to oxygen and nitrogen radical toxicity and have been shown to contract during cerebral ischemia and remain contracted despite reopening of the occluded artery. This causes impaired re-flow and may diminish the benefit of re-canalization therapies in stroke patients. Hyperglycemia-induced dysfunction of the signaling pathways between pericytes and endothelia is thought to play an important role in diabetic retinopathy, a common cause of blindness. Amyloid deposits detected within degenerating pericytes in the brains of patients with Alzheimer's disease suggest that pericyte dysfunction may play a role in cerebral hypoperfusion and impaired amyloid ß-peptide clearance in Alzheimer's disease. This exciting possibility may reveal a novel temporal sequence of events in chronic neurodegeneration, in which microvascular dysfunction due to pericyte degeneration initiates secondary neurodegenerative changes. Identification of molecular mechanisms by which pericytes regulate BBB integrity in inflammatory conditions as well as in vasogenic brain edema may lead to new treatments. Pericytes may also take part in tissue repair and vascularization after CNS injury. In conclusion, although the evidence is just emerging and mostly preliminary, disclosing pericytes' role in the pathophysiology of CNS diseases may yield exciting developments and novel treatments.

Updated review on the link between cortical spreading depression and headache disorders.

Introduction: Experimental animal studies have revealed mechanisms that link cortical spreading depression (CSD) to the trigeminal activation mediating lateralized headache. However, conventional CSD as seen in lissencephalic brain is insufficient to explain some clinical features of aura and migraine headache.Areas covered: The importance of CSD in headache development including dysfunction of the thalamocortical network, neuroinflammation, calcitonin gene-related peptide, transgenic models, and the role of CSD in migraine triggers, treatment options, neuromodulation, and future directions are reviewed.Expert opinion: The conventional understanding of CSD marching across the hemisphere is invalid in gyrencephalic brains. Thalamocortical dysfunction and interruption of functional cortical network systems by CSD may provide alternative explanations for clinical manifestations of migraine phases including aura. Not all drugs showing CSD blocking properties in lissencephalic brains have efficacy in migraine headache and monoclonal antibodies against CGRP ligand/receptors which are effective in migraine treatment, have no impact on aura in humans or CSD properties in rodents. Functional networks and molecular mechanisms mediating and amplifying the effects of limited CSD in migraine brain remain to be investigated to define new targets.

Pericyte morphology and function.

The proper delivery of blood is essential for healthy neuronal function. The anatomical substrate for this precise mechanism is the neurovascular unit, which is formed by neurons, glial cells, endothelia, smooth muscle cells, and pericytes. Based on their particular location on the vessel wall, morphology, and protein expression, pericytes have been proposed as cells capable of regulating capillary blood flow. Pericytes are located around the microvessels, wrapping them with their processes. Their morphology and protein expression substantially vary along the vascular tree. Their contractibility is mediated by a unique cytoskeleton organization formed by filaments of actin that allows pericyte deformability with the consequent mechanical force transferred to the extracellular matrix for changing the diameter. Pericyte ultrastructure is characterized by large mitochondria likely to provide energy to regulate intracellular calcium concentration and fuel contraction. Accordingly, pericytes with compromised energy show a sustained intracellular calcium increase that leads to persistent microvascular constriction. Pericyte morphology is highly plastic and adapted for varying contractile capability along the microvascular tree, making pericytes ideal cells to regulate the capillary blood flow in response to local neuronal activity. Besides the vascular regulation, pericytes also play a role in the maintenance of the blood-brain/retina barrier, neovascularization and angiogenesis, and leukocyte transmigration. Here, we review the morphological and functional features of the pericytes as well as potential specific markers for the study of pericytes in the brain and retina.

A nanomedicine transports a peptide caspase-3 inhibitor across the blood-brain barrier and provides neuroprotection.

Caspases play an important role as mediators of cell death in acute and chronic neurological disorders. Although peptide inhibitors of caspases provide neuroprotection, they have to be administered intracerebroventricularly because they cannot cross the blood-brain barrier (BBB). Herein, we present a nanocarrier system that can transfer chitosan nanospheres loaded with N-benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethyl ketone (Z-DEVD-FMK), a relatively specific caspase-3 inhibitor, across BBB. Caspase-3 was chosen as a pharmacological target because of its central role in cell death. Polyethylene glycol-coated nanospheres were conjugated to an anti-mouse transferrin receptor monoclonal antibody (TfRMAb) that selectively recognizes the TfR type 1 on the cerebral vasculature. We demonstrate with intravital microscopy that this nanomedicine is rapidly transported across the BBB without being measurably taken up by liver and spleen. Pre- or post-treatment (2 h) with intravenously injected Z-DEVD-FMK-loaded nanospheres dose dependently decreased the infarct volume, neurological deficit, and ischemia-induced caspase-3 activity in mice subjected to 2 h of MCA occlusion and 24 h of reperfusion, suggesting that they released an amount of peptide sufficient to inhibit caspase activity. Similarly, nanospheres inhibited physiological caspase-3 activity during development in the neonatal mouse cerebellum on postnatal day 17 after closure of the BBB. Neither nanospheres functionalized with TfRMAb but not loaded with Z-DEVD-FMK nor nanospheres lacking TfRMAb but loaded with Z-DEVD-FMK had any effect on either paradigm, suggesting that inhibition of caspase activity and subsequent neuroprotection were due to efficient penetration of the peptide into brain. Thus, chitosan nanospheres open new and exciting opportunities for brain delivery of biologically active peptides that are useful for the treatment of CNS disorders.

Post-stroke inflammatory response is linked to volume loss in the contralateral hemisphere.

OBJECTIVES: There is a delicate homeostatic balance between the central nervous system and immune system. Stroke triggers an immunodepressive state to suppress a potential immune reaction directed against neuroglial tissue; however, this supposedly protective response inadvertently results in an infection-prone, and thereby a pro-inflammatory setting. In this study, we assessed the magnitude of cerebral volume loss in the unaffected contralateral hemisphere following stroke, and determined its relationship with inflammatory cascades. METHODS: The volume of the hemisphere contralateral to the ischemic insult was measured on admission and follow-up MRI's in 50 ischemic stroke patients. Information related to clinical features, infectious complications, and markers of inflammation (erythrocyte sedimentation rate, neutrophil/lymphocyte ratio, C-reactive protein) were prospectively collected, and their relationship with hemispheric volume change was evaluated using bivariate and multivariate statistics. RESULTS: The contralateral hemisphere volume decreased by a median (interquartile range) of 14 (4-32) mL after a follow-up duration of 101 (63-123) days (p < .001); the volume reduction was 0.8 (0.2-1.8) % per month with respect to baseline. Old age, atrial fibrillation, stroke severity, C-reactive protein level, neutrophil/lymphocyte ratio, and development of infections during hospitalization were significantly associated with volume loss (p < .05). Stroke severity (NIHSS score or infarct volume) and inflammation related parameters (neutrophil/lymphocyte ratio or systemic infections) remained independently and positively associated with volume loss in multivariate regression models. CONCLUSIONS: Cerebral tissue changes following stroke are not limited to the ischemic hemisphere. Apart from stroke severity, a pro-inflammatory state and post-stroke infections contribute to cerebral volume loss in the non-ischemic hemisphere.

F-actin polymerization contributes to pericyte contractility in retinal capillaries.

Although it has been documented that central nervous system pericytes are able to contract in response to physiological, pharmacological or pathological stimuli, the underlying mechanism of pericyte contractility is incompletely understood especially in downstream pericytes that express low amounts of alpha-smooth muscle actin (α-SMA). To study whether pericyte contraction involves F-actin polymerization as in vascular smooth muscle cells, we increased retinal microvascular pericyte tonus by intravitreal injection of a vasoconstrictive agent, noradrenaline (NA). The contralateral eye of each mouse was used for vehicle injection. The retinas were rapidly extracted and fixed within 2 min after injections. Polymeric/filamentous (F-actin) and monomeric/globular (G-actin) forms of actin were labeled by fluorescently-conjugated phalloidin and deoxyribonuclease-I, respectively. We studied 108 and 83 pericytes from 6 NA- and 6 vehicle-treated retinas and, found that F/G-actin ratio, a microscopy-based index of F-actin polymerization, significantly increased in NA-treated retinas [median (IQR): 4.2 (3.1) vs. 3.5 (2.1), p = .006], suggesting a role for F-actin polymerization in pericyte contractility. Shift from G-actin monomers to polymerized F-actin was more pronounced in 5th and 6th order contracted pericytes compared to non-contracted ones [7.6 (4.7) vs. 3.2 (1.2), p < .001], possibly due to their dependence on de novo F-actin polymerization for contractile force generation because they express α-SMA in low quantities. Capillaries showing F-actin polymerization had significantly reduced diameters compared to the ones that did not exhibit increased F/G-actin ratio in pericytes [near soma / branch origin diameter; 0.67 (0.14) vs. 0.81 (0.34), p = .005]. NA-responsive capillaries generally did not show nodal constrictions but a tide-like diameter decrease, reaching a maximum near pericyte soma. These findings suggest that pericytes on high order downstream capillaries have F-actin-mediated contractile capability, which may contribute to the vascular resistance and blood flow regulation in capillary bed.

Hydrogen peroxide-induced stroke: elucidation of the mechanism in vivo.

OBJECT: Hydrogen peroxide (H2O2) is used as a hemostatic agent in many neurosurgery centers. The authors used a 3% H2O2 solution for final hemostasis after removal of a left insular tumor. Immediately afterward, air bubbles were observed within the lumen of the polar temporal artery. Postoperative MR imaging revealed punctate areas of infarction in the lenticulostriate artery territory. The authors designed an experimental study to elucidate the mechanism of remote O2 emboli and reactive O2 species-related vasoactive responses and thrombus formation. METHODS: In this study, H2O2 irrigation was used in mice with either an intact pial layer or after the pia mater was removed through a corticotomy. Normal saline irrigation was used in the corresponding control groups. Vessels were examined for intravascular O2 emboli under the microscope. Tissue sections were then obtained and stained with H & E and the 3-nitrotyrosine (3-NT) antibody to evaluate intravascular thrombus formation and peroxynitrite reaction, respectively. RESULTS: Multiple bubbles were observed within the lumen of the vessels after exposure to H2O2 regardless of whether the pial layer was destroyed or intact. Immunofluorescent staining for 3-NT showed an abundant positive reaction in the vessel walls of all animals exposed to H2O2 as well as vascular occlusion with acute thrombus formation. Samples taken from the animals that received saline showed no positive staining for 3-NT and no vascular occlusion. CONCLUSIONS: Exposure to H2O2 may cause serious ischemic complications. The formation of peroxynitrite may cause vasoactive responses to H2O2 and platelet aggregation/thrombus formation, and the free diffusion of H2O2 through the vessel walls and its conversion to water and O2 leads to O2 bubbles within the closed vessel lumen. If used intradurally, H2O2 may have deleterious ischemic effects, and it can only be used carefully in open extradural spaces.